Generation of inner ear cells

ABSTRACT

Methods for generating cells of the inner ear, e.g., hair cells and supporting cells, from stem cells, e.g., mesenchymal stem cells, are provided, as well as compositions including the inner ear cells. Methods for the therapeutic use of the inner ear cells for the treatment of hearing loss are also described.

CLAIM OF PRIORITY

This application is continuation application of U.S. patent applicationSer. No. 12/233,017, filed Sep. 18, 2008, which is a continuation ofInternational Patent Application No. PCT/US2007/084654, filed on Nov.14, 2007, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/859,041, filed on Nov. 15, 2006; the entirecontents of each of the foregoing applications are hereby incorporatedby reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. F33DC006789, RO1 DC007174, and P30 DC05209 from the National Institute onDeafness and other Communicative Disorders (NIDCD) of the NationalInstitutes of Health. The Government has certain rights in theinvention.

TECHNICAL FIELD

This invention relates to methods using bone marrow mesenchymal stemcells to regenerate inner ear cells, e.g., hair cells and supportingcells, to treat inner ear damage.

BACKGROUND

A source of sensory cells and neurons for regeneration of inner earcells would provide a valuable tool for clinical application becauseneurons and hair cells could be employed in cell replacement therapy forhearing loss. Recent work has shown that hair cells and neurons can bedifferentiated from endogenous stem cells of the inner ear (Li et al.,Nat Med 9, 1293-1299 (2003); Rask-Andersen et al., Hear Res 203, 180-191(2005)) and other work has shown that endogenous cells of the sensoryepithelium can be converted to hair cells when the proneuraltranscription factor, Atoh1, is expressed exogenously (Izumikawa et al.,Nat Med 11, 271-276 (2005); Zheng and Gao, Nat Neurosci 3, 580-586(2000)) and yet the endogenous stem cells of the inner ear do notspontaneously generate hair cells. Injection of whole bone marrow toreconstitute a lethally irradiated mouse resulted in engraftment ofthese cells in areas occupied by inner ear mesenchymal cells andfibrocytes but did not yield hair cells (Lang et al., J Comp Neurol 496,187-201 (2006)).

SUMMARY

The present invention is based, at least in part, on the discovery ofmethods that can be used to induce stem cells to differentiate into haircells and supporting cells. Thus, described herein are methods forproviding populations of hair cells and/or supporting cells,compositions comprising said cells, and methods of use thereof, e.g.,for the treatment of subjects who have or are at risk of developing ahearing loss.

In one aspect, the invention provides methods for providing populationsof hair cells and/or supporting cells. The methods include:

obtaining a population of stem cells with neurogenic potential;

culturing the stem cells under conditions sufficient to induce thedifferentiation of at least some of the stem cells into inner earprogenitor cells, and doing one (or more) of the following:

-   -   (i) inducing the expression of Atoh1 in the inner ear progenitor        cells, in an amount and for a time sufficient to induce at least        some of the inner ear progenitor cells to differentiate into        hair cells;    -   (ii) contacting the inner ear progenitor cells with an inhibitor        of Notch signalling (e.g., a gamma-secretase inhibitor or        inhibitory nucleic acid), in an amount and for a time sufficient        to induce at least some of the inner ear progenitor cells to        differentiate into hair cells; or    -   (iii) culturing the inner ear progenitor cells in the presence        of chick otocyst cells for a time and under conditions        sufficient for at least some of the inner ear progenitor cells        to differentiate into hair cells,        thereby providing populations of hair cells and/or supporting        cells.

In some embodiments, the methods include isolating the inner earprogenitor cells, hair cells, and/or supporting cells, e.g., to providea purified population thereof.

In some embodiments, the inner ear progenitor cells express nestin,sox2, musashi, Brn3C, Pax2, and Atoh1.

In some embodiments, the hair cells express one or more genes selectedfrom the group consisting of Atoh1, jagged 2, Brn3c, p27Kip, Ngn1,NeuroD, myosin VIIa and espin. In some embodiments, the hair cellsexpress jagged 2, Brn3c, myosin VIIa and espin. In some embodiments, thehair cells express F-actin in a V pattern on the apical surface of thecells.

In some embodiments, the supporting cells express one or more ofclaudin14, connexin 26, p75^(Trk), Notch 1, and S100A.

In some embodiments, the methods further include transplanting the haircells or supporting cells into a subject in need thereof, e.g., into ornear the sensory epithelium of the subject. In some embodiments, thepopulation of stem cells is obtained from a subject in need of thetransplant.

Also described herein are isolated populations of hair cells, supportingcells, and inner ear progenitor cells obtained by a method describedherein.

In another aspect, the invention features methods for treating a subjectwho has or is at risk for developing a disorder, e.g., a hearingdisorder or vestibular disorder, wherein the disorder is treatable witha transplant of hair cells and/or supporting cells, the methodcomprising transplanting cells obtained by a method described hereininto the cochlea of the subject, thereby treating the subject. In theseembodiments, it is preferable if the population of stem cells wasobtained from the subject in need of the transplant.

In some embodiments, inducing the expression of Atoh1 in the cellscomprises inducing the expression of exogenous Atoh1 in the cells, e.g.,by transducing the cells with a vector encoding a Atoh1 polypeptide,e.g., a plasmid vector or a viral vector, e.g., an adenovirus,lentivirus, or retrovirus.

In some embodiments, inducing the expression of exogenous Atoh1 in thestem cells comprises increasing expression of endogenous Atoh1, e.g., byincreasing activity of the Atoh1 promoter or by replacing the endogenousAtoh1 promoter with a more highly active promoter.

In some embodiments, culturing the stem cells in the presence of chickotocyst cells for a time and under conditions sufficient for at leastsome of the stem cells to differentiate into hair cells comprisesculturing the stem cells in medium comprising IGF, EGF, and FGF.

In some embodiments, the stem cells used in the methods described hereinare mesenchymal stem cells. In some embodiments, the stem cells used inthe methods described herein are human stem cells.

As noted, the invention also features cells isolated by a methoddescribed herein, as well as compositions containing them.

Methods for treating subjects (e.g., mammals such as humans) who have,or who are at risk for developing, a hearing loss, are also describedherein. These methods include administering a cell or population ofcells (as described herein; e.g., a population of hair cells obtained bydifferentiating a population of stem cells) to the ear of the patient,e.g., to the cochlea. The administered cells may be obtained by themethods described herein, and the starting material may be stem cellsobtained from the patient to be treated.

There may be certain advantages to the use of the cells described hereinfor the treatment of hearing loss. For example, the stem cells can beobtained from humans for clinical applications. Because the stem cellscan be harvested from a human, and in particular can be harvested fromthe human in need of treatment, the immunological hurdles common inxeno- and allotransplantation experiments can be largely avoided.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a row of four photomicrographs of bone marrow MSCs frompassage 3 immunostained with antibodies against CD44, CD45, CD34 andSca-1 followed by secondary antibodies against mouse immunoglobulinslabeled with TRITC (medium gray, shown in red in the original). Stainingfor CD34 and CD45 was negative, but CD44 and Sca-1 were expressed.Nuclei were stained with DAPI (darker gray, blue in the original).

FIG. 1B is a row of four photomicrographs of bone marrow MSCs frompassage 3 immunolabeled for CD44 (first panel, medium gray, shown in redin the original) and nestin (second panel, lighter gray, shown ingreen). The third panel is a DAPI nuclear stain (blue in original). Themerged image in the right-most panel shows co-staining of a populationof cells with both markers (lightest gray, yellow/orange in theoriginal)

FIG. 1C is a row of four photomicrographs of bone marrow MSCs frompassage 3 stained for co-expression of Sca-1 (first panel, red in theoriginal) and nestin (second panel, green in the original). Merged imagein the right-most panel shows co-staining.

FIG. 1D is a row of four plots showing the results of analysis of bonemarrow MSCs by chip flow cytometry indicating the ratio ofimmunopositive cells for each of the listed antibodies (CD44, firstpanel; Sca-1, second panel; CD34, third panel; and CD45, last panel);axes are “Fluorescence” and “No. of events.”

FIG. 1E is a pair of photomicrographs showing the potential for lineagedifferentiation, as demonstrated by formation of chondrocytes andextracellular matrix after treatment of bone marrow MSCs with TGF-β.Cells that grew out from a micro-aggregate (left) were stained for typeII collagen (right).

FIG. 1F is a pair of photomicrographs showing the differentiation ofbone marrow MSCs to neurons by differentiation in serum-free mediumcontaining neuronal growth supplements and bFGF. Staining forneurofilament (NF-M) is shown in these cells.

FIG. 2A is a gel showing the results of genetic analysis for neuralprogenitor markers by RT-PCR of MSCs treated with IGF-1, EGF and bFGFfor 14 days followed by analysis. MSC (bone marrow MSCs), NP (neuralprogenitors at 2 wks after induction of progenitor formation). The genesanalyzed are shown to the left of the gel.

FIGS. 2B-C are two sets of four photomicrographs showing that the neuralprogenitor marker, nestin, visualized by immunohistochemistry using asecondary antibody labeled with FITC (top right panel in 2B and 2C,shown in green in the original), was co-expressed with CD44 (2B, topleft panel, shown in red in the original) and with Sca-1 (2C, top leftpanel, shown in red in the original). DAPI is shown in blue (lower leftpanel in each figure). Scale bars are 50 μm. Merged images in the lowerright panel of each figure show coexpression of nestin and CD44 (2B) orSca1 (2C) (all of the cells appeared green in the original, indicatingcoexpression).

FIG. 3A is a gel showing the results of genetic analysis by RT-PCR ofprecursor cells incubated in NT3, FGF and BDNF (which support neuronaland sensory cell progenitors in the inner ear). The gene profilesincluded expression of Oct4, nestin, Otx2, and Musashi, as well asproneural transcription factors, GATA3, NeuroD, Ngn1, Atoh1, Brn3c, andZic2. These cells did not express hair cells genes, myosin VIIa andespin.

FIG. 3B is a gel showing the results of genetic analysis by RT-PCR ofthe cells obtained after induction with NT3, FGF, and BDNF. Genescharacteristic of supporting cells (claudin14, connexin 26, p75^(Trk),Notch 1, and S100A) were also observed. These progenitor cells thus hadexpression profiles characteristic of neuronal or sensory progenitors.Genes analyzed are shown to the left of the gels.

FIG. 4A is a photomicrograph showing exogenous expression of Atoh1 inbone marrow MSCs; expression was observed in cells and nuclei (green inthe original) due to the expression of GFP from the vector.

FIG. 4B is a gel showing the results of gene expression in cellstransfected with Atoh1 followed by treatment of the cells with NT3, FGFand BDNF. The results indicate that this protocol gave rise toprogenitor cells that subsequently matured into cells expressing haircell genes, including espin, myosin VIIa, jagged 2, and Brn3c, andp27Kip, in addition to the proneural genes, Ngn1 and NeuroD.

FIG. 4C is a gel showing the results of further genetic analysis of thecells under the differentiating conditions described in 4B; the resultsshowed that the cells also expressed S100A, p75^(Trk), claudin 14,connexin 26, and Notch1, consistent with some cells having a supportingcell phenotype.

FIG. 4D is a photomicrograph of an MSC cell line selected in Zeocin; thecells had a high percentage of GFP expression when cultured in serum(green in original).

FIG. 4E is a row of 4 photomicrographs of cells stained for Myo7a (firstpanel), Math1/Atoh1 (second panel), or DAPI (third panel); the lastpanel is a merged image. After differentiation, the number of haircell-like cells per DAPI nucleus rose and these cells stained for myosinVIIa (shown in red in the first panel) and Atoh1 (shown in green in thesecond panel; arrows in the second and last panels).

FIG. 4F is two rows of 4 photomicrographs of an Atoh1 expressing cellline differentiated to cells with nuclei that were immunopositive forBrn3c (second column, green in original; indicated by arrowheads) andcytoplasm positive for myosin VIIa (first column, red in original;indicated by arrows). Nuclei were stained with DAPI (third column, bluein original).

FIG. 4G is a row of three photomicrographs showing that thedifferentiated cells were positive for F-actin which protruded from theapex of the cell in the shape of a stereocilia bundle (arrow).

FIG. 4H is a row of three photomicrographs showing that F-actin stainingwas arranged in a characteristic V pattern on the apical surface.

FIG. 5A is a gel showing the results of genetic analysis of bone marrowMSC derived progenitors were co-cultured for 21 days with chick otocystcells that had been treated with mitomycin C (Mito C); the resultsshowed that expression of jagged 2, p27Kip, Atoh1, Brn3c, myosin VIIaand espin was increased, whereas the expression of these genes in chickcells was undetectable. Chick otocyst cells that had been fixed byincubation with paraformaldehyde were less effective (PFA) than theunfixed cells but did cause differentiation of the progenitors.Conditioned medium from the chick cells (Cnd Med) had no effect (levelsof expression of these markers similar to previously shown data fordifferentiating conditions).

FIG. 5B is a set of three photomicrographs showing that expression ofAtoh1 (Math-1, middle panel, green in original) and myosin VIIa (toppanel, red in original) in cells from a Atoh1-GFP mouse showed greenfluorescence corresponding to the induction of this marker in thenucleus and had expression of myosin VIIa in the cytoplasm.

FIG. 6A is a set of four photomicrographs showing an increase influorescence (green in original) indicating the conversion of bonemarrow cells to cells expressing Atoh1. The cells stained for Atoh1(Math1, bottom left, green in original), myosin VIIa (top left, red inoriginal) and DAPI (top right, blue in original). A merged image isshown in on the bottom right panel.

FIG. 6B is a photomicrograph showing that Atoh1-expressing cells werefound incorporated into the tissue of the chick otic epithelium. Thehair cells of the chick were stained with the chick-specific marker, HCA(white in original) and myosin VIIa (red in original), whereas theAtoh-1 expressing mouse cells were green due to expression of GFP(arrows).

FIG. 6C is a set of four photomicrographs showing a lack of cell fusion,demonstrated by the presence of HCA (arrowhead, lower panels) in cellsthat did not have green fluorescence and of Atoh1-GFP (arrow, rightcolumn) exclusively in cells that did not stain for HCA, a marker forchicken cells. No cells with both GFP and HCA were observed in theseexperiments. Scale bars are 100 μm.

FIG. 7 is a gel showing the results of genetic analysis of cells afterinhibition of Notch signaling with an inhibitor of γ-secretase increasesexpression of hair cell markers. Gene expression in MSCs treated with aγ-secretase inhibitor showed that loss of Notch signaling increasedAtoh1 expression. The timing of inhibition was critical: γ-secretaseinhibitor added at d1 of differentiation in vitro for a total of 10 daysled to an increase in hair cell markers, myosin VIIa and espin, whereasinhibitor added at d3 did not induce hair cell markers.

DETAILED DESCRIPTION

Although stem cells are present in the inner ear (Li et al., Trends MolMed 10, 309-315 (2004); Li et al., Nat Med 9, 1293-1299 (2003);Rask-Andersen et al., Hear Res 203, 180-191 (2005)), hair cells do notregenerate after damage, and, therefore, a source of cells that couldpotentially be used for cell transplantation in a therapeuticreplacement of these sensory cells has important implications fortreatment of sensorineural hearing loss. Bone marrow has been harvestedand used extensively in clinical applications and is a highly desirablesource, because cells from a patient's bone marrow could potentially betransplanted without the problem of immune rejection. The presentmethods include a treatment regimen for hearing loss includingtransplantation of hair cells obtained by methods described herein.

By a combination of growth factor stimulation and expression of thetranscription factor, Atoh1, that is required for hair cell formation inthe inner ear, the present inventors demonstrate herein that stem cells,e.g., mesenchymal stem cells derived from bone marrow, can be induced todifferentiate into hair cells. In addition, the neurosensory progenitorsobtained from bone marrow can be converted to sensory cells byco-culture with cells of the developing sensory epithelium, even in theabsence of Atoh1 expression.

Stem cells in bone marrow are known to be the precursors for alllymphoid and erythroid cells, but mesenchymal stem cells in bone marrowalso act as precursors to bone, cartilage, and fat cells (Colter et al.,Proc Natl Acad Sci USA 97, 3213-3218 (2000); Pittenger et al., Science284, 143-147 (1999)). In addition to mesenchymal tissues, these stemcells have been shown to give rise to cells of other lineages includingpancreatic cells (Hess et al., Nat Biotechnol 21, 763-770 (2003)),muscle cells (Doyonnas et al., Proc Natl Acad Sci USA 101, 13507-13512(2004)) and neurons (Dezawa et al., J Clin Invest 113, 1701-1710 (2004);Hermann et al., J Cell Sci 117, 4411-4422 (2004); Jiang et al., ProcNatl Acad Sci USA 100 Suppl 1, 11854-11860 (2003)). The evidenceprovided herein demonstrates an extended range of cell fates availablefor these bone marrow-derived cells that includes cells of theneurosensory lineage, even including differentiation to inner ear haircells.

Methods for Generating Cells of the Inner Ear

Methods of generating cells of the inner ear are provided, includingprogenitor cells and differentiated inner ear cells including hair cellsand supporting cells. Stem cells are unspecialized cells capable ofextensive proliferation. Stem cells are pluripotent and are believed tohave the capacity to differentiate into most cell types in the body(Pedersen, Scientif. Am. 280:68 (1999)), including neural cells, musclecells, blood cells, epithelial cells, skin cells, and cells of the innerear (e.g., hair cells and cells of the spiral ganglion). Stem cells arecapable of ongoing proliferation in vitro without differentiating. Asthey divide, they retain a normal karyotype, and they retain thecapacity to differentiate to produce adult cell types.

Hematopoietic stem cells resident in bone marrow are the source of bloodcells, but in addition to these hematopoietic stem cells, the bonemarrow contains mesenchymal stem cells (MSCs) that can differentiateinto cell types of all three embryonic germ layers (Colter et al., ProcNatl Acad Sci USA 97, 3213-3218 (2000); Doyonnas et al., Proc Natl AcadSci USA 101, 13507-13512 (2004); Herzog et al., Blood 102, 3483-3493(2003); Hess et al., Nat Biotechnol 21, 763-770 (2003); Jiang et al.,Nature 418, 41-49 (2002); Pittenger et al., Science 284, 143-147(1999)). This has been demonstrated in vivo in studies that tracktransplanted bone marrow cells to specific tissues where theydifferentiate into the resident tissue type (Mezey et al., Proc NatlAcad Sci USA 100, 1364-1369 (2003); Weimann et al., Proc Natl Acad SciUSA 100, 2088-2093 (2003)).

Many of these cells have been used for transplantation and are apreferred source of new cells for therapies because the transplantedcells are immunologically matched when harvested from a patient to betreated and because they have been extensively used in clinicalapplications so that their safety is known.

Stem cells can differentiate to varying degrees. For example, stem cellscan form cell aggregates called embryoid bodies in hanging dropcultures. The embryoid bodies contain neural progenitor cells that canbe selected by their expression of an early marker gene such as Sox1 andthe nestin gene, which encodes an intermediate filament protein (Lee etal., Nat. Biotech. 18:675-9, 2000).

Neurogenic Stem Cells

Inner ear cells or inner ear cell progenitors can be generated frommammalian stem cells. As described herein, stem cells suitable for usein the present methods can be any stem cell that has neurogenicpotential, i.e., any stem cell that has the potential to differentiateinto a neural cell, e.g., neurons, glia, astrocytes, retinalphotoreceptors, oligodendrocytes, olfactory cells, hair cells,supporting cells, and the like. Neurogenic stem cells, including humanadult stem cells such as bone marrow mesenchymal stem cells, can beinduced to differentiate into inner ear progenitor cells that arecapable of giving rise to mature inner ear cells including hair cellsand supporting cells. Neurogenic stem cells useful in the methodsdescribed herein can be identified by the expression of certainneurogenic stem cell markers, such as nestin, sox1, sox2, and musashi.Alternatively or in addition, these cells express high levels ofhelix-loop-helix transcription factors NeuroD, Atoh1, and neurogenin1.

Examples of neurogenic stem cells include embryonic stem cells or stemcells derived from mature (e.g., adult) tissue, such as the ear (e.g.,inner ear), central nervous system, blood, skin, eye or bone marrow. Insome embodiments, the stem cells are mesenchymal stem cells. Any of themethods described herein for culturing stem cells and inducingdifferentiation into inner ear cells (e.g., hair cells or supportingcells) can be used.

Stem cells useful for generating cells of the inner ear can be derivedfrom a mammal, such as a human, mouse, rat, pig, sheep, goat, ornon-human primate. For example, stem cells have been identified andisolated from the mouse utricular macula (Li et al., Nature Medicine9:1293-1299, 2003).

Generation of Neural Progenitor Cells

There are a number of induction protocols known in the art for inducingdifferentiation of stem cells with neurogenic potential into neuralprogenitor cells, including growth factor treatment (e.g., treatmentwith EGF, FGF, and IGF, as described herein) and neurotrophin treatment(e.g., treatment with NT3 and BDNF, as described herein). Otherdifferentiation protocols are known in the art; see, e.g., Corrales etal., J. Neurobiol. 66(13):1489-500 (2006); Kim et al., Nature 418, 50-6(2002); Lee et al., Nat Biotechnol 18, 675-9 (2000); and Li et al., NatBiotechnol 23, 215-21 (2005).

As one example of an induction protocol, the stem cells are grown in thepresence of supplemental growth factors that induce differentiation intoprogenitor cells. These supplemental growth factors are added to theculture medium. The type and concentration of the supplemental growthfactors is be adjusted to modulate the growth characteristics of thecells (e.g., to stimulate or sensitize the cells to differentiate) andto permit the survival of the differentiated cells such as neurons,glial cells, supporting cells or hair cells.

Exemplary supplementary growth factors are discussed in detail below,and include, but are not limited to basic fibroblast growth factor(bFGF), insulin-like growth factor (IGF), and epidermal growth factor(EGF). Alternatively, the supplemental growth factors can include theneurotrophic factors neurotrophin-3 (NT3) and brain derived neurotrophicfactor (BDNF). Concentrations of growth factors can range from about 100ng/mL to about 0.5 ng/mL (e.g., from about 80 ng/mL to about 3 ng/mL,such as about 60 ng/mL, about 50 ng/mL, about 40 ng/mL, about 30 ng/mL,about 20 ng/mL, about 10 ng/mL, or about 5 ng/mL).

Neural progenitor cells produced by these methods include inner earprogenitor cells, i.e., cells that can give rise to inner ear cells suchas hair cells and supporting cells. Inner ear progenitor cells can beidentified by the expression of marker genes such as nestin, sox2, andmusashi, in addition to certain inner-ear specific marker genes Brn3C,Pax2, and Atoh1. The invention includes purified populations of innerear progenitor cells expressing nestin, sox2, musashi, Brn3C, Pax2, andAtoh1. These inner ear progenitor cells are lineage committed, and canbe induced to further differentiate into hair cells and supporting cellsby a method described herein.

Progenitor cells prepared by a method described herein can optionally befrozen for future use.

Cell Culture Methods

In general, standard culture methods are used in the methods describedherein. Appropriate culture medium is described in the art, such as inLi et al. (supra). For example, stem cells can be cultured in serum freeDMEM/high-glucose and F12 media (mixed 1:1), and supplemented with N2and B27 solutions and growth factors. Growth factors such as EGF, IGF-1,and bFGF have been demonstrated to augment sphere formation in culture.In vitro, stem cells often show a distinct potential for forming spheresby proliferation of single cells. Thus, the identification and isolationof spheres can aid in the process of isolating stem cells from maturetissue for use in making differentiated cells of the inner ear. Thegrowth medium for cultured stem cells can contain one or more or anycombination of growth factors. This includes leukemia inhibitory factor(LIF) which prevents the stem cells from differentiating. To induce thecells (and the cells of the spheres) to differentiate, the medium can beexchanged for medium lacking growth factors. For example, the medium canbe serum-free DMEM/high glucose and F12 media (mixed 1:1) supplementedwith N2 and B27 solutions. Equivalent alternative media and nutrientscan also be used. Culture conditions can be optimized using methodsknown in the art.

Differentiation by Expression of Atoh1

As described herein, expression of Atoh1 in stem-cell derived progenitorcells was sufficient to drive them into adopting hair cell markers.Studies of Atoh1 expression in the ear have indicated that thishelix-loop-helix transcription factor occupies a key place in thehierarchy of inner ear transcription factors for differentiation of haircells.

Atoh1 nucleic acids and polypeptides are known in the art, and describedin, for example, U.S. Pat. Nos. 6,838,444 and 7,053,200, and P.G. PUB.Nos. 2004/0237127 and 2004/0231009, all to Zoghbi et al., allincorporated by reference in their entirety. In some embodiments, theAtoh1 is, or is at least 80%, 85%, 90%, 93%, or 95% identical to, humanatonal homolog 1 (ATOH1); ATH1; and HATH1 (for additional informationsee Ben-Arie et al., Molec. Genet. 5: 1207-1216 (1996); Bermingham etal., Science 284: 1837-1841 (1999); OMIM *601461; UniGene Hs.532680;GenBank Accession Nos. NM_(—)005172.1 (nucleic acid) and NP_(—)005163.1(polypeptide)). Other species can also be used, e.g., Mouse Atoh1 (alsoknown as Math1, GenBank Acc. No. NM_(—)007500.2), chicken Atoh1 (alsoknown as Cath1; GenBank Acc. No. AF467292.1).

The human Atoh1 mRNA (CDS=−1065) and polypeptide sequences are asfollows:

(SEQ ID NO: 1)   1 atgtcccgcc tgctgcatgc agaagagtgg gctgaagtga aggagttggg agaccaccat  61 cgccagcccc agccgcatca tctcccgcaa ccgccgccgc cgccgcagcc acctgcaact 121 ttgcaggcga gagagcatcc cgtctacccg cctgagctgt ccctcctgga cagcaccgac 181 ccacgcgcct ggctggctcc cactttgcag ggcatctgca cggcacgcgc cgcccagtat 241 ttgctacatt ccccggagct gggtgcctca gaggccgctg cgccccggga cgaggtggac 301 ggccgggggg agctggtaag gaggagcagc ggcggtgcca gcagcagcaa gagccccggg 361 ccggtgaaag tgcgggaaca gctgtgcaag ctgaaaggcg gggtggtggt agacgagctg 421 ggctgcagcc gccaacgggc cccttccagc aaacaggtga atggggtgca gaagcagaga 481 cggctagcag ccaacgccag ggagcggcgc aggatgcatg ggctgaacca cgccttcgac 541 cagctgcgca atgttatccc gtcgttcaac aacgacaaga agctgtccaa atatgagacc 601 ctgcagatgg cccaaatcta catcaacgcc ttgtccgagc tgctacaaac gcccagcgga 661 ggggaacagc caccgccgcc tccagcctcc tgcaaaagcg accaccacca ccttcgcacc 721 gcggcctcct atgaaggggg cgcgggcaac gcgaccgcag ctggggctca gcaggcttcc 781 ggagggagcc agcggccgac cccgcccggg agttgccgga ctcgcttctc agccccagct 841 tctgcgggag ggtactcggt gcagctggac gctctgcact tctcgacttt cgaggacagc 901 gccctgacag cgatgatggc gcaaaagaat ttgtctcctt ctctccccgg gagcatcttg 961 cagccagtgc aggaggaaaa cagcaaaact tcgcctcggt cccacagaag cgacggggaa1021 ttttcccccc attcccatta cagtgactcg gatgaggcaa gttag (SEQ ID NO: 2)MSRLLHAEEWAEVKELGDHHRQPQPHHLPQPPPPPQPPATLQAREHPVYPPELSLLDSTDPRAWLAPTLQGICTARAAQYLLHSPELGASEAAAPRDEVDGRGELVRRSSGGASSSKSPGPVKVREQLCKLKGGVVVDELGCSRQRAPSSKQVNGVQKQRRLAANARERRRMHGLNHAFDQLRNVIPSFNNDKKLSKYETLQMAQIYINALSELLQTPSGGEQPPPPPASCKSDHHHLRTAASYEGGAGNATAAGAQQASGGSQRPTPPGSCRTRFSAPASAGGYSVQLDALHFSTFEDSALTAMMAQKNLSPSLPGSILQPVQEENSKTSPRSHRSDGEFSPHSHYSDS DEAS

The mouse Atoh1 mRNA (CDS=196-1251) and polypeptide sequences are asfollows:

(SEQ ID NO: 3)   1 tcgacccacg cgtccgccca cgcgtccgga tctccgagtg agagggggag ggtcagagga  61 ggaaggaaaa aaaaatcaga ccttgcagaa gagactagga aggtttttgt tgttgttgtt 121 cggggcttat ccccttcgtt gaactgggtt gccagcacct cctctaacac ggcacctccg 181 agccattgca gtgcgatgtc ccgcctgctg catgcagaag agtgggctga ggtaaaagag 241 ttgggggacc accatcgcca tccccagccg caccacgtcc cgccgctgac gccacagcca 301 cctgctaccc tgcaggcgag agaccttccc gtctacccgg cagaactgtc cctcctggat 361 agcaccgacc cacgcgcctg gctgactccc actttgcagg gcctctgcac ggcacgcgcc 421 gcccagtatc tgctgcattc tcccgagctg ggtgcctccg aggccgcggc gccccgggac 481 gaggctgaca gccagggtga gctggtaagg agaagcggct gtggcggcct cagcaagagc 541 cccgggcccg tcaaagtacg ggaacagctg tgcaagctga agggtggggt tgtagtggac 601 gagcttggct gcagccgcca gcgagcccct tccagcaaac aggtgaatgg ggtacagaag 661 caaaggaggc tggcagcaaa cgcaagggaa cggcgcagga tgcacgggct gaaccacgcc 721 ttcgaccagc tgcgcaacgt tatcccgtcc ttcaacaacg acaagaagct gtccaaatat 781 gagaccctac agatggccca gatctacatc aacgctctgt cggagttgct gcagactccc 841 aatgtcggag agcaaccgcc gccgcccaca gcttcctgca aaaatgacca ccatcacctt 901 cgcaccgcct cctcctatga aggaggtgcg ggcgcctctg cggtagctgg ggctcagcca 961 gccccgggag ggggcccgag acctaccccg cccgggcctt gccggactcg cttctcaggc1021 ccagcttcct ctgggggtta ctcggtgcag ctggacgctt tgcacttccc agccttcgag1081 gacagggccc taacagcgat gatggcacag aaggacctgt cgccttcgct gcccgggggc1141 atcctgcagc ctgtacagga ggacaacagc aaaacatctc ccagatccca cagaagtgac1201 ggagagtttt ccccccactc tcattacagt gactctgatg aggccagtta ggaaggcaac1261 agctccctga aaactgagac aaccaaatgc ccttcctagc gcgcgggaag ccccgtgaca1321 aatatccctg caccctttaa tttttggtct gtggtgatcg ttgttagcaa cgacttgact1381 tcggacggct gcagctcttc caatcccctt cctcctacct tctccttcct ctgtatgtag1441 atactgtatc attatatgta cctttacgtg gcatcgtttc atggtccatg ctgccaatat1501 gctgctaaaa tgtcgtatct ctgcctctgg tctgggtttc acttatttta taccttggga1561 gttcatcctt gcgtgttgcg ctcactcaca aataagggag ttagtcaatg aagttgtttc1621 cccaactgct tgagacccgc attgggtact ttactgaaca cggactattg tgttgttaaa1681 atgcaggggc agataagagt atctgtagag cttagacacc aagtgtgtcc agcagtgtgt1741 ctagcggacc cagaatacac gcacttcatc actggccgct gcgccgcctt gaagaaactc1801 aactgccaat gcagagcaac ttttgatttt aaaaacagcc actcataatc attaaactct1861 ttgcaaatgt ttgtttttgc aaatgaaaat taaaaaaaaa catgtagtgt caaaggcatt1921 tggtcaattt tattttgctt tgttaacatt agaaaagtta tttattattg cgtatttgga1981 cccatttcta cttaattgcc ttttttttac attttctact cgagatcgtt ttattttgat2041 ttagcaaatc cagttgccat tgctttatgt atgtatgctc ttttacaaat gataaaataa2101 actcggaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa (SEQ ID NO: 4)MSRLLHAEEWAEVKELGDHHRHPQPHHVPPLTPQPPATLQARDLLVRRSGCGGLSKSPGPVKVREQLCKLKGGVVVDELGCSRQRAPSSKQVNGVQKQRRLAANARERRRMHGLNHAFDQLRNVIPSFNNDKKLSKYETLQMAQIYINALSELLQTPNVGASSGGYSVQLDALHFPAFEDRALTAMMAQKDLSPSLPGGILQPVQEDNSKTSPRSHRSDGEFSPHSHYSDSDEAS

The chicken Cath1mRNA (CDS=1-717) and polypeptide sequences are asfollows:

(SEQ ID NO: 5)  1 atggccccag gaggtagcga gtgttgttgc agtgatgccg cgcacatcac ttggaggcag 61 tgggagtaca cgcacgagaa ccaactgtgc gtggcaggaa ctgtcagcag gatgaggccc121 aggacgtggg tctgcaccgg atctttgtgg gaccaggaag cgggaattac tttgatgggc181 ccccaaatac ccaaagtgga tgaggcagga gtgatgaccc acccggcaag gtcgctttgc241 agcactgggg cacatccgtg tcccggggtg gtcgtgctgc ccacgggtgg gatagggcag301 ccttcaaaga agctctccaa gtacgagacg ctgcagatgg cgcaaatcta catcagcgcc361 ctcgccgagc ttctgcacgg gccgcccgcg ccccccgagc cgcccgccaa ggccgagctc421 cgcggggccc ccttcgagcc tcccccgccg ccccctcctc cgccgccccg cgcctcgccc481 cccgcgcccg ccaggactcg cttccccccg gcggcggccg cgggcggttt cgcggcgctt541 ctcgagccgc tgcgcttccc ttctttcccg gcgcagaaag cgccttctcc cgcgctgctc601 ctggggccgc ccgcgccgca gcagcccgag aggagcaaag cgtcgccgcg ctctcaccgc661 agcgacgggg agttctcgcc gcgctcccac tacagtgact cggacgaggc cagctag(SEQ ID NO: 6) MAPGGSECCCSDAAHITWRQWEYTHENQLCVAGTVSRMRPRTWVCTGSLWDQEAGITLMGPQIPKVDEAGVMTHPARSLCSTGAHPCPGVVVLPTGGIGQPSKKLSKYETLQMAQIYISALAELLHGPPAPPEPPAKAELRGAPFEPPPPPPPPPPRASPPAPARTRFPPAAAAGGFAALLEPLRFPSFPAQKAPSPALLLGPPAPQQPERSKASPRSHRSDGE FSPRSHYSDSDEAS

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 80% of the length of the reference sequence, and in someembodiments is at least 90% or 100%. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

For purposes of the present invention, the comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a Blossum 62 scoring matrix with a gap penalty of 12,a gap extend penalty of 4, and a frameshift gap penalty of 5.

In some embodiments, the methods include expressing in the cells a Atoh1polypeptide encoded by a nucleic acid that hybridizes to the human Atoh1mRNA under stringent conditions. As used herein, the term “stringentconditions” describes conditions for hybridization and washing.Stringent conditions as used herein are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. See,e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(2006).

In some embodiments, the methods include expressing exogenous Atoh1 in astem cell. This can be achieved, for example, by introducing anexpression vector in the cell. As used herein, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked and can include a plasmid, cosmid or viralvector. The vector can be capable of autonomous replication or it canintegrate into a host DNA. Viral vectors include, e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses.

A vector can include a Atoh1 nucleic acid in a form suitable forexpression of the nucleic acid in a host cell. Generally, the expressionvector includes one or more regulatory sequences operatively linked tothe nucleic acid sequence to be expressed. The term “regulatorysequence” includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Regulatory sequences includethose which direct constitutive expression of a nucleotide sequence, aswell as tissue-specific regulatory and/or inducible sequences. Thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like. The expression vectors can be introduced intohost cells using methods known in the art, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. See, e.g., CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (2006).

In the present methods, the Atoh1 polypeptide expressed in the stemcells will have the ability to induce differentiation of mesenchymalstem cells to hair cells and/or supporting cells, as described herein.

Differentiation by Culturing with Chick Otocysts

Also as described herein, the stem cell-derived progenitor cells alsoresponded to physical contact with developing otocyst cells from thechicken embryo by differentiating into sensory epithelial cells, withoutthe requirement for exogenous Atoh1. This was evidenced by nGFPexpression from a Atoh1 enhancer-GFP reporter construct andco-expression of myosin VIIa after co-culture and differentiation, asdescribed herein. Neurons that express markers of sensory cells havebeen induced from bone marrow MSCs in previous work by incubation withotocyst and hindbrain-conditioned medium (Kondo et al., Proc Natl AcadSci USA 102, 4789-4794 (2005)) from embryonic mice.

Thus, the methods described herein can include contacting progenitorcells with otocyst cells, e.g., cells isolated from E3 embryonic chicks,as described herein.

In some embodiments, the methods include culturing the progenitor cellswith the otocyst cells in a ratio of about 50,000 cells per confluentlayer of otocyst cells, or by injection of 100,000 cells into an intactotocyst (see Examples, below). Alternatively, the stem cells can becultured in the presence of chick otocyst-conditioned media, which canbe produced using methods known in the art, e.g., using media that hasbeen in contact with a culture of chick otocysts for at about four days.

Differentiation by Inhibition of Notch Signalling

Notch is a plasma membrane receptor, and the Notch pathway consists ofNotch and its ligands, as well as intracellular proteins that transmitthe Notch signal to the nucleus. Included in the Notch pathway are thetranscription factors that bear the effector function of the pathway.

Notch signaling plays a role in lateral inhibition, in which one cell issingled out from a cell cluster for a given fate (e.g., differentiationinto a hair cell, for example). Differentiation is inhibited in thosecells not selected to differentiate, resulting in the prevention of aspecified fate commitment on the part of most of the cells of a cluster.Lateral inhibition occurs repeatedly during development. Central to thisprocess is binding to the Notch receptor of one of several ligands,including Delta, Scabrous and Serrate. Ligand binding to Notch ligandtriggers a chain of intracellular events resulting in lateralinhibition. A review of the Notch pathway can be found atArtavanis-Tsakonas et al., Science 268: 225-232 (1995). As describedherein, inhibition of Notch in the inner ear progenitor cells describedherein results in differentiation of the cells into hair cells andsupporting cells.

Thus, in some embodiments of the methods described herein, progenitorcells are grown in the presence of a Notch signalling pathway inhibitor.Exemplary Notch pathway inhibitors include γ-secretase inhibitors, ofwhich a number are known in the art (e.g., arylsulfonamides (AS),dibenzazepines (DBZ), benzodiazepines (BZ),N—[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester(DAPT), L-685,458 (Sigma-Aldrich), and MK0752 (Merck). A usefulconcentration will depend on the inhibitor chosen.

Other Notch inhibitors include inhibitory nucleic acids (e.g., smallinterfering RNAs, antisense oligonucleotides, and morpholino oligos;methods for designing, making, and using them are known in the art,e.g., gene walk methods for selecting and optimizing inhibitorysequences, see, e.g., Engelke, RNA Interference (RNAi): The Nuts & Boltsof siRNA Technology, (DNA Press, 2004); Mol, Antisense Nucleic Acids andProteins, (CRC, 1994); Sioud, Ribozymes and Sirna Protocols (Methods inMolecular Biology), (Humana Press; 2nd edition 2004); and Philips,Antisense Therapeutics (Methods in Molecular Medicine), (Humana Press2004)) targeting Notch (see, e.g., Presente et al., Proc. Nat. Acad.Sci. 101(6):1764-1768 (2004); Ivanov et al., Proc. Nat. Acad. Sci.101(46):16216-16221 (2004)) or its ligands, i.e., Delta or Jagged (see,e.g., Patzel et al., Nature Biotechnology 23, 1440-1444 (2005); Purow etal., Cancer Research 65:2353-2363 (2005); or Stallwood et al., J.Immunol. 177:885-895 (2006)). Alternatively, the cells can be modifiedto express m-Numb (GenBank Acc. No. NP_(—)001005743.1) or disheveled(Dvl; the human homologs are at GenBank Acc. No. NM_(—)004421.2 (variant1); NM_(—)004422.2 (variant 2); and NM_(—)004423.3 (variant 3), bothendogenous inhibitors of Notch signalling.

Assaying Differentiation

A variety of methods can be utilized to determine that a stem cell hasdifferentiated into a progenitor cell, or into a cell of the inner ear,e.g., a hair cell or supporting cell. For example, the cell can beexamined for the expression of a cell marker gene. Hair cell markergenes include myosin VIIa (myoVIIa), Atoh1, α9 acetylcholine receptor,espin, parvalbumin 3, and Brn3c. Supporting cell markers includeclaudin14, connexin 26, p75Trk, Notch 1, and S100A. Pluripotent stemcells generally do not express these genes. A stem cell that propagatesand produces a cell expressing one or more of these genes, has produceda hair cell, i.e., the stem cell has differentiated at least partiallyinto a hair cell. A stem cell that has differentiated into an inner earprogenitor cell (a precursor of hair cells) expresses early ear markergenes such as nestin, sox2, musashi, Brn3C, Pax2, and Atoh1. Aprogenitor cell can express one or more of these genes. The progenitorcells can be propagated in serum-free medium in the presence of growthfactors. Removal of growth factors and expression of Atoh1, orco-culture with chick otocysts, will induce the cells to differentiatefurther, such as into hair cells and supporting cells.

Identification of a hair cell or hair cell progenitor (e.g., a haircell, supporting cell, or progenitor cell that differentiated from astem cell) can be facilitated by the detection of expression of markergenes as described herein. Detection of the products of gene expressioncan be by immunocytochemistry. Immunocytochemistry techniques involvethe staining of cells or tissues using antibodies against theappropriate antigen. In this case, the appropriate antigen is theprotein product of the tissue-specific gene expression. Although, inprinciple, a first antibody (i.e., the antibody that binds the antigen)can be labeled, it is more common (and improves the visualization) touse a second antibody directed against the first (e.g., an anti-IgG).This second antibody is conjugated either with fluorochromes, orappropriate enzymes for colorimetric reactions, or gold beads (forelectron microscopy), or with the biotin-avidin system, so that thelocation of the primary antibody, and thus the antigen, can berecognized. The protein marker can also be detected by flow cytometryusing antibodies against these antigens, or by Western blot analysis ofcell extracts.

Alternatively or in addition, gene expression can be analyzed directly,e.g., using PCR methods known in the art, including quantitative PCR,e.g., quantitative RT-PCR, which can be used to detect and comparelevels of expression.

Methods of Treatment

The methods described herein can be used to generate cells fortherapeutic use. Treatment methods include generating cells of the innerear (e.g., hair cells or supporting cells) from stem cells, using amethod described herein, for transplantation into an ear of a human inneed thereof. Transplantation of the cells into the inner ear of asubject can be useful for restoring or improving the ability of thesubject to hear, or for decreasing the symptoms of vestibulardysfunction. Inner ear cells derived from stem cells according to themethods described herein need not be fully differentiated to betherapeutically useful. A partially differentiated cell that improvesany symptom of a hearing disorder in a subject is useful for thetherapeutic compositions and methods described herein.

A human having a disorder of the inner ear, or at risk for developingsuch a disorder, can be treated with inner ear cells (hair cells orsupporting cells) generated from stem cells using a method describedherein. In a successful engraftment, at least some transplanted haircells, for example, will form synaptic contacts with spiral ganglioncells, and integrate into the sensory epithelium of the inner ear. Toimprove the ability of the cells to engraft, the stem cells can bemodified prior to differentiation. For example, the cells can beengineered to overexpress one or more anti-apoptotic genes in theprogenitor or differentiated cells. The Fak tyrosine kinase or Akt genesare candidate anti-apoptotic genes that can be useful for this purpose;overexpression of FAK or Akt can prevent cell death in spiral ganglioncells and encourage engraftment when transplanted into another tissue,such as an explanted organ of Corti (see for example, Mangi et al., Nat.Med. 9:1195-201 (2003)). Neural progenitor cells overexpressing α_(v)β₃integrin may have an enhanced ability to extend neurites into a tissueexplant, as the integrin has been shown to mediate neurite extensionfrom spiral ganglion neurons on laminin substrates (Aletsee et al.,Audiol. Neurootol. 6:57-65 (2001)). In another example, ephrinB2 andephrinB3 expression can be altered, such as by silencing with RNAi oroverexpression with an exogenously expressed cDNA, to modify EphA4signaling events. Spiral ganglion neurons have been shown to be guidedby signals from EphA4 that are mediated by cell surface expression ofephrin-B2 and -B3 (Brors et al., J. Comp. Neurol. 462:90-100 (2003)).Inactivation of this guidance signal may enhance the number of neuronsthat reach their target in an adult inner ear. Exogenous factors such asthe neurotrophins BDNF and NT3, and LIF can be added to tissuetransplants to enhance the extension of neurites and their growthtowards a target tissue in vivo and in ex vivo tissue cultures. Neuriteextension of sensory neurons can be enhanced by the addition ofneurotrophins (BDNF, NT3) and LIF (Gillespie et al., NeuroReport12:275-279 (2001)). A Sonic hedgehog (Shh) polypeptide or polypeptidefragment (e.g., SHH-N), can also be useful as an endogenous factor toenhance neurite extension. Shh is a developmental modulator for theinner ear and a chemoattractant for axons (Charron et al., Cell 113:1123 (2003)).

Any human experiencing or at risk for developing a hearing loss is acandidate for the treatment methods described herein. For example, thehuman can receive a transplant of inner ear hair cells or supportingcells generated by a method described herein. A human having or at riskfor developing a hearing loss can hear less well than the average humanbeing, or less well than a human before experiencing the hearing loss.For example, hearing can be diminished by at least 5, 10, 30, 50% ormore. The human can have sensorineural hearing loss, which results fromdamage or malfunction of the sensory part (the cochlea) or the neuralpart (the auditory nerve) of the ear, or conductive hearing loss, whichis caused by blockage or damage in the outer and/or middle ear, or thehuman can have mixed hearing loss, which is caused by a problem in boththe conductive pathway (in the outer or middle ear) and in the nervepathway (the inner ear). An example of a mixed hearing loss is aconductive loss due to a middle-ear infection combined with asensorineural loss due to damage associated with aging.

The subject can be deaf or have a hearing loss for any reason or as aresult of any type of event. For example, a human can be deaf because ofa genetic or congenital defect; for example, a human can have been deafsince birth, or can be deaf or hard-of-hearing as a result of a gradualloss of hearing due to a genetic or congenital defect. In anotherexample, a human can be deaf or hard-of-hearing as a result of atraumatic event, such as a physical trauma to a structure of the ear, ora sudden loud noise, or a prolonged exposure to loud noises. Forexample, prolonged exposures to concert venues, airport runways, andconstruction areas can cause inner ear damage and subsequent hearingloss. A human can experience chemical-induced ototoxicity, whereinototoxins include therapeutic drugs including antineoplastic agents,salicylates, quinines, and aminoglycoside antibiotics, contaminants infoods or medicinals, and environmental or industrial pollutants. A humancan have a hearing disorder that results from aging, or the human canhave tinnitus (characterized by ringing in the ears).

The cells can be administered by any suitable method. For example, torestore hearing, inner ear cells generated by a method described hereincan be transplanted, such as in the form of a cell suspension, into theear by injection, such as into the luminae of the cochlea. See, e.g.,the methods described in Corrales et al., J. Neurobiol. 66(13):1489-500(2006) and Hu et al., Experimental Cell Research 302:40-47 (2005).Injection can be, for example, through the round window of the ear orthrough the bony capsule surrounding the cochlea. The cells can beinjected through the round window into the auditory nerve trunk in theinternal auditory meatus or into the scala tympani. In a preferredembodiment, the cells are administered into or near the sensoryepithelium of the subject, e.g., into a fluid (perilymph)-filled spaceabove or below the sensory epithelium, i.e., the scala media, scalatympani, or scala vestibuli.

Alternatively, a human suitable for the therapeutic compositions andmethods described herein can include a human having a vestibulardysfunction, including bilateral and unilateral vestibular dysfunction.Vestibular dysfunction is an inner ear dysfunction characterized bysymptoms that include dizziness, imbalance, vertigo, nausea, and fuzzyvision and may be accompanied by hearing problems, fatigue and changesin cognitive functioning. Vestibular dysfunction can be the result of agenetic or congenital defect; an infection, such as a viral or bacterialinfection; or an injury, such as a traumatic or nontraumatic injury.Vestibular dysfunction is most commonly tested by measuring individualsymptoms of the disorder (e.g., vertigo, nausea, and fuzzy vision). Inthese embodiments, the inner ear cells generated by a method describedherein can be transplanted, such as in the form of a cell suspension,e.g., by injection, into an organ of the vestibular system, e.g., theutricle, ampulla and sacculus. The cells would generally be injectedinto the perilymph of these organs or into the vestibule (which connectsthe 3 organs).

Following treatment with an inner ear cell or inner ear cell progenitoras described herein, the human can be tested for an improvement inhearing or in other symptoms related to inner ear disorders. Methods formeasuring hearing are well-known and include pure tone audiometry, airconduction, and bone conduction tests. These exams measure the limits ofloudness (intensity) and pitch (frequency) that a human can hear.Hearing tests in humans include behavioral observation audiometry (forinfants to seven months), visual reinforcement orientation audiometry(for children 7 months to 3 years) and play audiometry for childrenolder than 3 years. Oto-acoustic emission testing can be used to testthe functioning of the cochlear hair cells, and electro-cochleographyprovides information about the functioning of the cochlea and the firstpart of the nerve pathway to the brain.

The therapeutic compositions and methods described herein can be usedprophylactically, such as to prevent hearing loss, deafness, or otherauditory disorder associated with loss of inner ear function. Forexample, a composition containing a differentiation agent can beadministered with a second therapeutic, such as a therapeutic that mayeffect a hearing disorder. Such ototoxic drugs include the antibioticsneomycin, kanamycin, amikacin, viomycin, gentamycin, tobramycin,erythromycin, vancomycin, and streptomycin; chemotherapeutics such ascisplatin; nonsteroidal anti-inflammatory drugs (NSAIDs) such as cholinemagnesium trisalicylate, diclofenac, diflunisal, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate,nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, salsalate,sulindac, and tolmetin; diuretics; salicylates such as aspirin; andcertain malaria treatments such as quinine and chloroquine.

For example, a human undergoing chemotherapy can also be administered aninner ear cell or inner ear cell progenitor as described herein, by amethod described herein. The chemotherapeutic agent cisplatin, forexample, is known to cause hearing loss. Therefore, a compositioncontaining a differentiation agent can be administered with cisplatintherapy to prevent or lessen the severity of the cisplatin side effect.An inner ear cell or inner ear cell progenitor as described herein canbe administered before, after and/or simultaneously with the secondtherapeutic agent. The two treatments generally will be administered bydifferent routes of administration.

The compositions and methods featured in the invention are appropriatefor the treatment of hearing disorders resulting from sensorineural haircell loss or auditory neuropathy. For example, patients withsensorineural hair cell loss experience the degeneration of cochlearhair cells, which frequently results in the loss of spiral ganglionneurons in regions of hair cell loss, and may also experience loss ofsupporting cells in the organ of Corti, and degeneration of the limbus,spiral ligament, and stria vascularis in the temporal bone material.Such patients may benefit particularly from administration of supportingcells and/or hair cells into the inner ear.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Sensory Progenitors from Mesenchymal Stem Cells

Mesenchymal stem cells were obtained from mouse bone marrow by culturingadherent cells from the marrow under high serum conditions.

Briefly, cells were obtained from bilateral femurs and tibias of 4 weekold C57BL/6 or Atoh1-nGFP mice (Helms et al., Development 127, 1185-1196(2000)) by flushing out the bone marrow with MEM-α (Gibco/BRL)containing 10% fetal bovine serum (FBS; BioWhittaker, Cambrex, N.Y.) and1 mM glutamine (Gibco/BRL). Pelleted cells were resuspended and mixedwith RBC lysis buffer (Gibco/BRL). Approximately 5×10⁶ cells werecultured on a 10 cm dish overnight in MEM-α with 9% horse serum, 9% FBS,1% Gluta-Max (Invitrogen) and 100 units/ml penicillin and streptomycin(100 μg/ml, Sigma) at 37° C. in a 5% CO₂ atmosphere. Nonadherenthematopoietic stem cells were removed, leaving adherent bone marrowstromal cells. When the cells became confluent, trypsinization wasperformed and the cells were cultured and passaged three to five times,with media changes every 3-4 days. These cells are referred to asmesenchymal stem cells (MSC).

Immunohistochemistry was performed as follows. Cells were fixed for 10min with 4% paraformaldehyde in PBS. Immunostaining was initiated byrehydrating and blocking the sections for 1 h with 0.1% Triton X-100 inPBS supplemented with 1% BSA and 5% goat serum (PBT1). Fixed andpermeabilized cells or rehydrated sections were incubated overnight inPBT1. CD34, CD44, CD45, Sca-1 antibodies (BD Biosciences) diluted 1:40were used for the characterization of extracted bone marrow cells. Haircells and bone marrow progenitors were characterized using monoclonalantibody to chick hair cell specific antigen diluted 1:500 (gift fromGuy Richardson (Bartolami et al., J Comp Neurol 314, 777-788 (1991));polyclonal antibody to myosin VIIa, 1:500 (Oshima et al., J Assoc ResOtolaryngol. 8(1):18-31 (2007)); monoclonal antibody to nestin, 1,000(Developmental Studies Hybridoma Bank, Iowa City, Iowa); polyclonalantibody to parvalbumin 3, 1:2,000 (Heller et al., J Assoc ResOtolaryngol 3, 488-498 (2002)); monoclonal antibody to Atoh1, 1:100(Developmental Studies Hybridoma Bank); monoclonal antibody toneurofilament M, 1:200 (Chemicon); Polyclonal antibody to collagen typeII, 1:40 (Chemicon); polyclonal antibody to Brn3c (Covance, Princeton);Cy-5 conjugated F-actin 1:1000 (Molecular probe). Samples were washedthree times for 20 min each with PBS. Anti-rabbit, anti-guinea pig andanti-mouse secondary antibodies conjugated with FITC-, TRITC-, andCy-5-(Jackson ImmunoResearch) were used to detect primary antibodies.The samples were counterstained with DAPI for 10 min (VectorLaboratories) and viewed by epifluorescence microscopy (Axioskop 2 MotAxiocam, Zeiss) or confocal microscopy (TCS, Leica). The counting ofimmunopositive cells was performed by counting 300 cells in 20 randomlyselected microscopic fields and significance was calculated by Student'st-test.

Flow cytometric analysis was also performed. MSC were incubated withantibodies to CD34, CD44, CD45 or Sca-1 (BD Biosciences) and furtherincubated with secondary anti-mouse antibody conjugated to TRITC. Datawere acquired and analyzed using an Agilent 2100 Bioanalyzer system andflow cytometry chips (Agilent Technology Inc., Palo Alto, Calif.). Thereference window was set so that fluorescence from the secondaryantibody alone was less than 2%.

The MSCs were negative for CD34 and CD45, markers for hematopoietic stemcells in bone marrow (Jiang et al., Nature 418, 41-49 (2002); Pittengeret al., Science 284, 143-147 (1999)) and positive for CD44 and Sca-1,markers for MSCs (Dezawa et al., J Clin Invest 113, 1701-1710 (2004)).Sca-1 was present on 5.2% of the cells and CD44 was present on 11.5% ofthe cells based on immunohistochemistry and the percentages determinedby flow cytometry were similar (FIGS. 1A and 1D and Table 1). Wedetected co-expression of CD44 and nestin as well as Sca-1 and nestin ona small percentage of the cells (FIGS. 1B and 1C).

TABLE 1 Co-Expression of CD44 and Sca-1 with Nestin in Mesenchymal StemCells pre-induction (%) post-induction (%) Nestin (+) cells 4.7 ± 0.814.2 ± 2.0  CD44 (+) cells 11.5 ± 1.6  11.9 ± 1.8  Sca-1 (+) cells 5.2 ±1.5 5.0 ± 0.4 CD 44 & nestin (+) cells 3.4 ± 0.9 9.9 ± 0.9 Sca-1 &nestin (+) cells 2.8 ± 1.2 4.3 ± 0.5 Positive cells were counted inrelation to total nuclei stained by DAPI. Data are mean ± SE for 10separate experiments. The increase in cells staining with nestin wassignificant (p < 0.001) as was the increase in the cells staining forboth nestin and CD44 (p < 0.001) and nestin and Sca-1 (p < 0.05).

We confirmed the previously reported capacity of MSCs to be converted tochondrocytes (Pittenger et al., Science 284, 143-147 (1999)) and neurons(Dezawa et al., J Clin Invest 113, 1701-1710 (2004)). For chondrogenicdifferentiation, MSC were formed into a micropellet and cultured in DMEMwith 10 ng/ml TGFbeta1, 6.25 ug/ml transferrin and 6.25 ug/ml insulinfor 2 weeks. Their potential to differentiate into chondrocytes isdemonstrated in FIG. 1E. For neuronal differentiation, MSC were culturedin DMEM/F12 1:1 containing N2/B27 supplement with bFGF (10 ng/ml) for 14days and for 7 days without FGF. This resulted in differentiation toneurons (Dezawa et al., J Clin Invest 113, 1701-1710 (2004)) as shown byneuronal markers (FIG. 1F).

To determine whether otic vesicle growth factors that are important inthe early development of inner ear progenitor cells could have a similareffect on MSCs, we removed the serum from the MSCs after 3-5 passagesand cultured the cells in serum-free medium containing IGF-1, EGF andbFGF.

For the induction of progenitor cells, passage 3-5 MSC were trypsinizedand transferred to 6-well plates or 4 well plates (BD Bioscience) coatedwith poly-L-ornithine and gelatin or fibronectin (Sigma) at 5×10⁴cells/ml. Cells were cultured for 5-7 days, and then cultured inserum-free medium composed of DMEM/F12 1:1 containing N2/B27 supplements(Invitrogen). For progenitor cell induction, we used a combination ofEGF (20 ng/ml) and IGF (50 ng/ml; R&D Systems, Minneapolis, Minn.) for 2weeks followed by the addition of bFGF (10 ng/ml) plus the other growthfactors for an additional 2 weeks, or a combination of NT3 (30 ng/ml)and bFGF (10 ng/ml) for 4-5 days followed by NT3 (30 ng/ml) and BDNF (10ng/ml) for 7 days.

Semiquantitative RT-PCR was performed as follows. Total RNA wasextracted with the RNAeasy minikit (Qiagen, Valencia, Calif.) accordingto the manufacturer's instructions. For reverse transcription, 6 μg oftotal RNA was used with SuperScript III transcriptase (Invitrogen) andoligo-dT primers. The PCR cycling conditions were optimized in pilotexperiments. Specific cycling parameters were: initial denaturation stepat 94° C. for 2 minutes, denaturation 94° C. for 30 seconds, annealingtemperature optimized between 56-60° C. for 30 seconds, extension 72° C.for 60 seconds, extension 72° C. for 60 seconds, and followed by 7minutes of terminal extension at 72° C. after the last cycle. The numberof cycles was optimized between 30 and 35, and conditions were keptconstant for each primer. The presented data are from experimentsrepeated at least 5 times. Control PCR without reverse transcriptase didnot produce specific bands. The primer pairs and cDNA product lengthswere as follows:

TABLE 2 RT-PCR—Primer Pairs and cDNA Product Length Forward ExpectedcDNA target primer SEQ ID NO: Reverse primer SEQ ID NO: product lengthOct4 ATG GCT 7. TTA ACC 8. 1033 bp GGA CAC CCA AAG CTG GCT CTC CAG TCA GGTT C Otx2 CCA TGA 9. GAA GCT 10. 211 bp CCT ATA CCA TAT CTC AGG CCC TGGCTT CAG G GTG GAA AG Sox2 CAC CCG 11. TCC CCT TCT 12. 414 bp GGC CTCCCA GTT AAC GCT CGC AGT CAC G CCA Pax2 CCA AAG 13. GGA TAG 14. 544 bpTGG TGG GAA GGA ACA AGA CGC TCA TTG CC AAG AC Pax6 AGA CTT 15. TAG CCA16. 589 bp TAA CCA GGT TGC AGG GCG GT GAA GAA CT Nestin AAC AGA 17.CTT CAG 18. 392 bp GAT TGG AAA GGC AAG GCC TGT CAC GCT GGC AGG AGMusashi ATG GAG 19. ATC TTC TTC 20. 332 bp ACT GAC GTC CGA GCG CCCGTG AC CAG GATA3 CCT CCG 21. ACC GTA 22. 319 bp ACG GCA GCC CTG GGA GTCACG GAG TTT Math1 AGA TCT 23. ACT GGC 24. 449 bp ACA TCA CTC ATC ACG CTCAGA GTC TGT C ACT G Neurogenin-1 TGG TGT 25. AAG GCC 26. 400 bp CGT CGGGAC CTC GGA AC CAA ACC TC NeuroD ACG GGC 27. TGA AAG 28. 513 bp TGA ACGAGA AGT CGG CGC TGC CAT TGG AC TGA TG Brn3c GCC ATG 29. ATG GCG 30.714 bp CGC CGA CCT AGA GTT TGT C TGA TGC Espin CAG CCT 31. TGA CCT 32.475 bp GAG TCA GTC GCT CCG CAG GCC AGG CCT C GCG CG Myo7a CTC CCT 33.AAG CAC 34. 628 bp CTA CAT CTG CTC CGC TCT CTG CTC GTT CG GTC CAC G Zic1GGC CAA 35. GAG AGC 36. 425 bp CCC CAA TGG GGT AAA GTC GCG TGT GTG AGG AZic2 GGC GGC 37. TTG CCA 38. 405 bp GCA GCT CAG CCC CCA CAA GGG AAACCA GTA GGA CAG TrkB TTG CCC 39. CGC TTG 40. 46 bp CTT CCC CTT CTC GCTTTA T CTC GT TrkC ACC CGC 41. TCC CGG 42. 521 bp ATC CCA TGT ACA GTC ATAAG TGC P27Kip CTG GAG 43. CGT CTG 44. 525 BP CGG ATG CTC CAC GAC GCCAGT GCC AGA C AGC Jag2 GTC CTT 45. GTT TCC 46. CCC ACA ACC TTGTGG GAG TT ACC TCG GT Notch1 AGA GAT 47. CAC ACA 48. 306 BP GTG GGAGGG AAC TGC AGG AC TTC ACC CT P75 GTC GTG 49. CTG TGA 50. GGC CTTGTT CAC GTG GCC ACT GGG G S100 GCC AAC 51. ACG TCG 52. 423 bp CGT GTGAGA CTG CTG CTG GGC AAG G Cla14 CCA GCA 53. GGG GCA 54. 664 bp CAG CGGCGG TTG TCC AG TCC TTG TAG Con26 CGG AAC 55. CTA AGC 56. 824 bp CAG AGAACG GGT TAG GAC TGC CTC CTA C ATC C Gapdh AAC GGG 57. CAG CCT 58. 442 bpAAG CCC TGG CAG ATC ACC CAC CAG

When the expression of neural progenitor cell markers in the resultingcultures was assessed, Otx2, nestin, Sox2, and Musashi were expressed inincreased amounts in these cells, which are subsequently referred toherein as progenitor cells, relative to MSCs based on RT-PCR (FIG. 2A).Pax6 was found in the progenitor cells but not in the MSCs (FIG. 2A).Pax2 was not expressed. A low level of Pax5 was detected but Pax8 wasnot expressed (data not shown). A similar pattern of expression was seenfor the stem cell marker, Oct4, which was expressed in the progenitorcells but interestingly, given its role in maintaining the pluripotencyof stem cells, was not found in the MSCs. The increase in expression ofnestin in the progenitor cells relative to the MSCs (FIG. 2A) wasconfirmed by immunohistochemistry (FIGS. 2B and 2C and Table 1) and wassignificant (p<0.001). Additional markers of the hair cell and neurallineages (Atoh1, Brn3c, GATA3) and neuronal markers (TrkB and TrkC) werealso expressed in the progenitors (FIG. 2A).

Because of the expression of TrkB and TrkC in the progenitor cellpopulations, we tested whether incubation with NT-3 and BDNF, theneurotrophins that bind to these receptors, would increase the yield ofprogenitor cells or alter the expression of genes for hair cell orneuronal fate. We found an increase in expression of Otx2, Sox2, nestin,and Musashi under these conditions as well as an increase in Oct4expression (FIG. 3A), indicating that the cells may have adopted aneural progenitor cell fate. The neurotrophin-mediated conversion toprogenitor cells had a more rapid time course that we found for EGF,IGF-1 and bFGF alone. The expression of proneural transcription factors,NeuroD and Ngn1, as well as neural and hair cell lineage markers, GATA3,Atoh1, and Brn3c, were also increased and the expression of Ngn1 andNeuroD, which select for a neural over a hair cell fate in the inner ear(Kim et al., Development 128, 417-426 (2001); Matei et al., Dev Dyn.234(3):633-50 (2005)) were higher when NT-3 and BDNF were included inthe differentiation medium. Other transcription factors expressed in theotic precursors during development, Zic2 and Pax6, were elevated in theprogenitor cells relative to the MSCs, and Zic1 expression was notobserved. This suggests that NT-3 and BDNF induced the formation ofcells of a neural lineage that were potentially destined to become bothneurons and hair cells. However, the cells were not converted to haircells or neurons because markers for these cells were not found (FIG.3A, hair cell markers myosin VIIa and espin). We also tested for theexpression of genes characteristic of other epithelial cells in thecochlea such as supporting cells, because the progenitors for hair cellscan include or give rise to these cells and found that the progenitorsexpressed S100A, p75^(trk), claudin 14, connexin 26, and Notch1 .

The observation of supporting cell markers from the MSC-derivedprogenitor cells after growth factor induction may be correlated totheir origin from a common progenitor during in vivo development (Mateiet al., Dev Dyn. 234(3):633-50 (2005); Satoh and Fekete, Development132, 1687-1697 (2005)). Since hair cells can be induced to develop fromsupporting cells after introduction of the Atoh1 gene (Izumikawa et al.,Nat Med 11, 271-276 (2005); Zheng and Gao, Nat Neurosci 3, 580-586(2000)), the role of supporting cells as potential progenitors for haircells via transdifferentiation has been discussed (Izumikawa et al., NatMed 11, 271-276 (2005)). The expression of supporting cell genes mayreflect an intermediate or accompanying stage on the way to becominghair cells; in Atoh1 knockout mice undifferentiated cells with markersof supporting cells have been observed to activate the Atoh1 gene(Fritzsch et al., Dev Dyn 233, 570-583 (2005); Woods et al., NatNeurosci 7, 1310-1318 (2004)). Alternatively, supporting cells could beinduced by the developing hair cells: ectopic hair cells in the greaterepithelial ridge induced supporting cell markers in surrounding cells(Woods et al., Nat Neurosci 7, 1310-1318 (2004)). The MSCs could beinduced to become hair cell progenitors by bFGF, EGF and IGF-1, factorsthat potentially stimulate the in vivo formation of these progenitors(Leon et al., Endocrinology 136, 3494-3503 (1995); Pauley et al., DevDyn 227, 203-215 (2003); Zheng et al., J Neurosci 17, 216-226 (1997)),and these progenitors were able to give rise to hair cells afteroverexpression of Atoh1. An increase in expression of neural progenitormarkers could be caused by expansion of the cells that express thesemarkers or by differentiation of MSCs to the neural progenitorphenotype.

As described herein, MSC-derived progenitor cells expressed neurotrophinreceptors. BDNF and NT-3 play important roles in maturation of inner earneurons (Fritzsch et al., J Neurosci 17, 6213-6225 (1997); Pirvola andYlikoski, Curr Top Dev Biol 57, 207-223 (2003)), and in differentiationof neural stem cells to neurons (Ito et al., Neurosci Res 71, 648-658(2003)), and we therefore tested whether the fate of the progenitorscould be modulated by neurotrophins Incubation with these factorsresulted in enrichment of progenitors that could be converted to haircells by subsequent Atoh1 overexpression (Izumikawa et al., Nat Med 11,271-276 (2005); Zheng and Gao, Nat Neurosci 3, 580-586 (2000)) orco-culture with chick otocyst cells. Since NT-3 and BDNF were found toincrease both Atoh1 expression and differentiation in neural stem cells(Ito et al., J Neurosci Res 71, 648-658 (2003)), neurotrophins coulddirectly increase differentiation of MSCs or could increase theircompetence to respond to overexpressed Atoh1 .

Analysis of the progenitor cells obtained from the MSCs revealedparallels with natural development of the inner ear sensory epithelia.The MSC-derived progenitors expressed Sox2, which must be present forsubsequent hair cell differentiation in the developing otocyst (Kiernanet al., Nature 434, 1031-1035 (2005)). The expression of Atoh1 in cellsthat did not have myosin VIIa and the appearance of myosin VIIa at latertime points is consistent with the order of their expression duringdevelopment based on immunohistochemistry (Chen et al., Development 129,2495-2505 (2002)). The lack of Pax2 expression was surprising since thepaired box transcription factor is ubiquitously expressed in the otocyst(Burton et al., Dev Biol 272, 161-175 (2004); Li et al., J Neurobiol 60,61-70 (2004)). This may suggest that Pax2 is not required or that it canbe replaced by another factor for the conversion of MSCs to hair cells.Pax5 was detected and may substitute for Pax2 based on their functionalequivalence (Bouchard et al., Development. 127(5):1017-28 (2000)). Thisis consistent with the analysis of the Pax2 null mouse (Burton et al.,Dev Biol 272, 161-175 (2004)), which appears to develop hair cellsdespite severe disruption of the normal morphology of the cochlea. Thelack of Zic1 expression relative to Zic2 is also found duringdevelopment of a hair cell phenotype as compared to sensory neurons inthe otocyst (Warner et al., Dev Dyn 226, 702-712 (2003)) and is thusconsistent with the development of a hair cell phenotype. Theidentification of inductive molecules on chick otocyst cells that arenot present in conditioned media will provide further insights into haircell differentiation.

The isolation of progenitor cells that can give rise to the tissue oforigin, as observed in the inner ear (Li et al., Trends Mol Med 10,309-315 (2004); Li et al., Nat Med 9, 1293-1299 (2003a)), might bepredicted and yet the cells do not regenerate after damage, possiblybecause of the decrease in number of inner ear stem cells after birth(Oshima et al., J Assoc Res Otolaryngol. 8(1):18-31 (2007)). Therefore,a source of cells to provide replacements for these sensory cells ishighly desirable. The in vivo role of MSCs in regeneration generallyremains uncertain although bone marrow could act as a source of newcells in organs with few progenitors. Despite the demonstration thatcells from bone marrow migrate into the brain and heart in adults(Oshima et al., J Assoc Res Otolaryngol. 8(1):18-31 (2007)); Weimann etal., Proc Natl Acad Sci USA 100, 2088-2093 (2003)) and differentiateinto neurons in the brain, hematopoietic stem cells from bone marrowwere not converted to cardiomyocytes after injection (Murry et al.,Nature 428, 664-668 (2004)) and conversion to neurons was extremely rare(Wagers et al., Science 297, 2256-2259 (2002); Weimann et al., Proc NatlAcad Sci USA 100, 2088-2093 (2003)). The most successful attempts atregeneration by adult stem cells from other tissues have been obtainedafter a lesion Doyonnas et al., Proc Natl Acad Sci USA 101, 13507-13512(2004); Edge, Transplant Proc 32, 1169-1171 (2000); Hess et al., NatBiotechnol 21, 763-770 (2003); Pagani et al., J Am Coll Cardiol 41,879-888 (2003)) and tissue damage may be required to see cellreplacement by bone marrow-derived cells. Whether bone marrow-derivedcells play any regenerative role in the sensory or peripheral nervoussystem in a spontaneous response to damage in vivo is an unansweredquestion, but, although low-level replacement of hair cells by bonemarrow cells in vivo cannot be ruled out, spontaneous replacement ofsensory cells is unlikely to be significant given the lack of hair cellregeneration seen in the adult cochlear and vestibular systems (Hawkinsand Lovett, Hum Mol Genet 13(Spec No 2):R289-296 (2004); White et al.,Nature 441, 984-987 (2006)).

Example 2 Transfection with an Atoh1 Expression Plasmid ConvertsProgenitors to Hair Cells

To test whether the progenitor cells could act as inner ear precursorcells, it was evaluated whether overexpression of Atoh1, a transcriptionfactor that is known to push competent progenitors to a hair cell fate(Izumikawa et al., Nat Med 11, 271-276 (2005); Zheng and Gao, NatNeurosci 3, 580-586 (2000)), would increase the expression of hair cellmarkers.

The efficiency of Atoh1 transfection was tested by counting greenfluorescent cells after transfection with a vector coding for GFPexpression in addition to Atoh1. We constructed a vector containing theAtoh1 coding sequence under EF1alpha-promotor control in the pTracer-EFvector (Invitrogen) that has a GFP-Zeocin fusion sequence under the CMVpromoter. Gene transfection was done in the progenitor cell state or asMSC using LIPOFECTAMINE™ transfection reagent (Sigma). Cells werecultured in Zeocin (Invitrogen) to obtain stable transfectants.Transfected MSC were cultured in the serum-free conditions withcombinations of growth factors.

When MSCs were transfected, as many as 2% of the cells were GFP positiveat 24 hours (FIG. 4A). RT-PCR at day 14 showed that the transfected cellpopulation expressed markers of developing sensory epithelia, such asp27Kip, Brn3c and jagged2, and mature hair cells markers, myosin VIIaand espin (FIG. 4B) as well as increased expression of Ngn1 and NeuroD.We also detected expression of supporting cell markers, S100A,p75^(Trk), claudin 14, connexin 26, and Notch1, indicating that theprogenitor cells could give rise to hair cells and supporting cells(FIG. 4C). Selection of MSC transfectants with stable Atoh1 expressionincreased the percentage of GFP-positive cells (FIG. 4D). Incubation ofthese cells in the growth factors described above followed byimmunohistochemistry yielded cells with expression of Atoh1 and myosinVIIa respectively in 7.7% and 7.1% of the total cells (FIG. 4E).Differentiation under growth factor stimulation gave rise to cells withBrn3c in the nucleus and myosin VIIa in the cytoplasm (FIG. 4F). Thesecells were positive for both markers in the same cells, with 92% of theAtoh1-positive cells showing staining for myosin VIIa, and 77% of theBrn3c-positive cells showing staining for myosin VIIa. Examination ofthe myosin VIIa positive cells for F-actin (FIGS. 4G and H) indicatedthat some of the cells (4.9% of the myosin VIIa-positive cells) haddeveloped protrusions at their apical poles. These protrusion had thepolarized appearance of stereociliary bundles and were positive forespin (FIG. 4G).

Atoh1 expression led to strong expression of helix-loop-helixtranscription factors, Ngn1 and NeuroD. Several previous studies haveindicated that Atoh1 expression can increase these transcriptionfactors. In mouse cerebellum Atoh1 expression leads to overexpression ofNeuroD (Helms et al., Mol Cell Neurosci 17, 671-682 (2001)). Inzebrafish NeuroD is not expressed in the absence of Atoh1 (Sarrazin etal., Dev Biol 295, 534-545 (2006)) and is required for hair cellformation. The related mouse achaete-scute (Mash1) upregulates Ngn1 (Cauet al., Development 124, 1611-1621 (1997)). However, Ngn1 wasdownregulated by overexpression of Atoh1 in chick neural tube (Gowan etal., Neuron 31, 219-232 (2001)).

These data demonstrate that overexpression of Atoh1 in growth-factorinduced progenitor cells induces the differentiation of a percentage ofthose cells to hair cells.

Example 3 Conversion of Progenitors to Hair Cells is Stimulated byDeveloping Otocyst Cells

To test whether the developing otocyst produced factors that wouldincrease the differentiation of MSCs to hair cells, co-cultureexperiments of E3 chick otocyst cells with MSCs were performed.

Embryos of the white leghorn strain (Charles River) were harvested 72hours after placing fertilized eggs onto rocking platforms in ahumidified incubator maintained at 38° C. The dissection of otocystsfrom the extracted embryos was done in cooled PBS, pH 7.2, after removalof periotic mesenchymal tissues. The otocysts were trypsinized anddissociated to single cells for plating and 2×10⁴ cells were culturedovernight in four-well plates in 10% FBS. One day after plating, theotocyst cells were fixed with 4% paraformaldehyde for 20 minutes, orinactivated with mitomicin C (10 μg/ml) for 3 hours, then washed 4 timeswith PBS. Conditioned medium from the cultured cells was collected andfrozen prior to use on progenitors cells. Progenitor cells (5×10⁴cells/ml) induced in serum-free medium with growth factors, wereoverlaid on the chick otocyst cells and cultured for 5-7 days withEGF/IGF, followed by 10 days with EGF/IGF/FGF and withdrawal of growthfactors for 5-10 more days. The cells were analyzed by RT-PCR orimmunohistochemistry as described herein.

After culture in the presence of the chick otocyst cells for 21 days,increased expression of myosin VIIa, jagged2, p27Kip, Brn3c and Atoh1 byRT-PCR was found (FIG. 5A). The factor(s) was unlikely to be a secretedmolecule because fixation of the cells did not diminish their ability topromote differentiation after exposure for 14 days, while conditionedmedium was ineffective in 14 days (FIG. 5A). Conversion of the stemcells to hair cells could be followed by appearance of greenfluorescence in the cultures using MSCs derived from transgenicAtoh1-nGFP mice that express a nuclear version of enhanced GFP whenAtoh1 enhancer elements are activated (Chen et al., Development 129,2495-2505 (2002); Lumpkin et al., Gene Expr Patterns 3, 389-395 (2003)).These green cells were observed in the co-cultures with chick otocystcells (FIG. 5B) and the cells were co-labeled with antibody to myosinVIIa.

The otocyst from E3 chick embryos were used for injection of progenitorcells. The dissected otocysts were transferred into 7 ml of serum-freeDMEM/F12 1:1 containing N2 and B27 on a gelatin-coated tissue culturedish. After attachment of intact otocysts, progenitor cells from MSC(5×10⁷ cells/ml) were injected into the otocyst with a micropipette in 2μl of medium. The left otic vesicles did not receive cell grafts andserved as controls. The otocysts were harvested after 10-14 days, fixed30 min in paraformaldehyde (4% in PBS), cryoprotected overnight insucrose (30% in PBS), embedded in TissueTek (EMS) and serially sectioned(16 μm) with a cryostat (CM3050, Leica, Nussloch, Germany).

When the progenitor cells were injected into chick otocysts obtained atE3, conversion of progenitors to cells with hair cell properties (5% ofthe myosin VIIa-positive cells were positive for nGFP) was observed(FIG. 6A). The murine hair cells were seen to incorporate into the haircell bearing epithelia of the developing chicken otocyst as detected byexpression of GFP (FIG. 6B). One possible explanation for the expressionof hair cell genes by the MSC-derived cells in co-culture is fusion withchick cells. To rule this out we labeled the cells with an antibody tochick hair cell antigen (Bartolami et al., J Comp Neurol 314, 777-788(1991)). Native chick hair cells could be detected lining the internalcavity of the otocyst (51% of 1,352 cells from 15 otocyst injectionsthat stained for myosin VIIa were positive for chick hair cell antigen),and the cells that expressed nGFP and hair cell markers did notco-express chick hair cell antigen (FIG. 6C) and were therefore of mouseorigin and not the product of cell fusion.

These experiments, performed in an attempt to understand how contact ofthe MSCs with developing otocyst cells provided a signal that inducedtheir differentiation to hair cells, demonstrated that the inductiveeffect was through a cell surface molecule as opposed to a secretedfactor. Injection of the MSC into the developing otocyst in vitroindicated that hair cells that differentiated from the stem cells wereintegrated into the chick otocyst epithelium, demonstrating that theenvironment provided by developing chicken otocyst cells could guidedifferentiation and integration of suitable progenitor cells. Theinstructive influence has also been seen previously with innerear-derived stem cells and murine ES cell-derived progenitor cells (Liet al., Trends Mol Med 10, 309-315 (2004); Li et al., Nat Med 9,1293-1299 (2003); Li et al., Proc Natl Acad Sci USA 100, 13495-13500(2003).

The effect of the co-incubation with otocyst cells may be simply toactivate Atoh1 expression and a sufficient amount of Atoh1 may berequired to allow hair cell differentiation since the MSCs had lowlevels of Atoh1 but did not have detectable sensory epithelial cellmarkers. This type of high level expression could be needed for Atoh1 toovercome the level of preexisting endogenous inhibitors that interactwith Atoh1 protein. The murine cells could be clearly distinguished fromthe chick hair cells that differentiated at the same time by theirexpression of nGFP and by immunolabeling of the chick hair cells with aspecies-specific antibody. The cells were never co-stained (based onexamination of 1,352 cells), indicating that the mouse hair cells haddifferentiated from stem cells and did not arise from cell fusion.

Example 4 Inhibition of Notch Signaling Induces Differentiation of HairCells

The Notch pathway maintains the alternating pattern of hair cells andsupporting cells in vivo by suppressing the differentiation of haircells from supporting cells and activation of Notch in the embryoappears to block development of hair cells from progenitors.

To examine the effect of the Notch pathway on the differentiation ofhair cells, the NT3/BDNF treated progenitors were incubated with aγ-secretase inhibitor. Analysis of gene expression in the progenitorsmade by incubation with NT3, BDNF, FGF and subsequently treated with theγ-secretase inhibitor demonstrated that loss of the notch signalingincreased Atoh1 expression. Atoh1 levels rose compared to the treatmentwith growth factors alone based on RT-PCR when the inhibitor was used at1 μM (FIG. 7). The timing of the addition of the inhibitor was essentialwith inhibition at a later stage (after 3 days of differentiation invitro) causing less induction of hair cell markers than inhibitionstarting at day 0 and continuing for 10 days. At the low concentration,γ-secretase inhibitor activates ngn1 and NeuroD and causes no increasein Atoh1 or hair cell markers. At higher concentration, the γ-secretaseinhibitor increases Atoh1 and Brn3c expression. The increased Atoh1appeared to be able to produce hair cells as the cells expressed markersfor the hair cells such as myosin7a, p27Kip. As HLH transcriptionfactors mediate the effects of the Notch pathway, this result isconsistent with the role of Notch and suggests a mechanism forpreventing hair cell differentiation under normal conditions.

Example 5 Inhibition of Hair Cell Differentiation in Human Stem Cells

To determine if human mesenchymal stem cells (hMSCs) can bedifferentiated into inner ear cell types including hair cells or sensoryneurons, human bone marrow cells from healthy adults were evaluated.

The human bone marrow cells were harvested and cultured as plated ontissue culture plastic for 16 hours, and nonadherent hematopoietic stemcells were aspirated.

First, the adherent cells were cultured in αMEM containing 9% horseserum and 9% fetal bovine serum and were negative for blood-forming cellmarkers, CD34 and CD45. These cells gave rise to chondrocytes expressingtype II and IV collagen after culture in the presence of TGFβ,transferrin and insulin.

Culture of hMSCs in DMEM/F12 medium containing N2 and B27 without serumin the presence of NT-3, BDNF, Sonic hedgehog and retinoic acid for 10days gave rise to cells that expressed neurosensory progenitor markersdetected by RT-PCR, Musashi, nestin, Pax6, Brn3a, NeuroD, Ngn1, andGATA3, and sensory neuron markers, peripherin and TrkC. Thesedifferentiated hMSCs were positive for β-III tubulin (2.1% of the totalcells were positive based on immunohistochemistry) and, of these cells,28% co-stained for peripherin and 31% co-stained for Brn3a.

For the differentiation to hair cells, hMSCs were transfected with humanAtoh1 in an expression vector with a selectable marker for eukaryoticcells. The selected progenitor cells expressed Atoh1 and, afterdifferentiation in DMEM/F12 medium containing N2 and B27 with NT-3 andBDNF for 10 days, expressed hair cell markers, Atoh1, myosin VIIa,p27Kip, Jag2 and espin based on RT-PCR.

The ability of these cells to engraft in an organ of Corti, theAtoh1-transfected cells were co-cultured with an ex vivo organ of Cortifrom mouse. This gave rise to cells expressing myosin VIIa and espinthat were detected by immunostaining, i.e., differentiated hair cells.When the ex vivo mouse organ of Corti was treated with toxins to inducehair cell degeneration, co-cultured bone marrow-derived cells wereobserved to engraft in the mouse sensory epithelium, thus demonstratingthe ability of cells obtained.

Thus, human MSCs are a potential alternative for cell-based treatment ofhearing loss, as they can be differentiated into inner ear cell typesincluding hair cells or sensory neurons, and can be successfullyengrafted into structures of the inner ear.

Example 6 Math1-Estrogen Receptor (ER) Fusion Constructs

One alternative to constitutive expression of Math1 is to use aconditional or inducible system of gene expression, to upregulate Math1with an inducer that is added to the cell medium or cochlearenvironment. An inducible model is particularly useful wheninvestigating the temporal effects of gene expression.

This Example describes a system in which administration of tamoxifen, asynthetic estrogen agonist, induces expression of Math1. AMath1-estrogen receptor (ER) fusion protein, where the ER has beenmutated so that it selectively binds to tamoxifen rather than estrogen,is constitutively expressed. In the absence of tamoxifen, the Math1-ERconstruct remains quiescent within the cytosol where it is inactivatedby heat shock proteins. The addition of tamoxifen to the transfectedcells results in a dose-dependent localization of the Math1-ER constructto the nucleus where it is transcribed leading to increased expressionof Math1. The sequence of Math1 is given above.

The sequence of ER used is as follows (SEQ ID NO:59):

ATGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGATGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGCCAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGTGGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGAAGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAACTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCTGCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGAAAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAACGCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCAGGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTATAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGGGAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGTGTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTTCCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAGAAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCCCTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGACTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGCCGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCTGGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAGTGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGGGATCAGGCTGGTGCCATGGGCGATCCACGAAATGAAATGGGTGCTTCAGGAGACATGAGGGCTGCCAACCTTTGGCCAAGCCCTCTTGTGATTAAGCACACTAAGAAGAATAGCCCTGCCTTGTCCTTGACAGCTGACCAGATGGTCAGTGCCTTGTTGGATGCTGAACCGCCCATGATCTATTCTGAATATGATCCTTCTAGACCCTTCAGTGAAGCCTCAATGATGGGCTTATTGACCAACCTAGCAGATAGGGAGCTGGTTCATATGATCAACTGGGCAAAGAGAGTGCCAGGCTTTGGGGACTTGAATCTCCATGATCAGGTCCACCTTCTCGAGTGTGCCTGGCTGGAGATTCTGATGATTGGTCTCGTCTGGCGCTCCATGGAACACCCGGGGAAGCTCCTGTTTGCTCCTAACTTGCTCCTGGACAGGAATCAAGGTAAATGTGTGGAAGGCATGGTGGAGATCTTTGACATGTTGCTTGCTACGTCAAGTCGGTTCCGCATGATGAACCTGCAGGGTGAAGAGTTTGTGTGCCTCAAATCCATCATTTTGCTTAATTCCGGAGTGTACACGTTTCTGTCCAGCACCTTGAAGTCTCTGGAAGAGAAGGACCACATCCACCGTGTCCTGGACAAGATCACAGACACTTTGATCCACCTGATGGCCAAAGCTGGCCTGACTCTGCAGCAGCAGCATCGCCGCCTAGCTCAGCTCCTTCTCATTCTTTCCCATATCCGGCACATGAGTAACAAACGCATGGAGCATCTCTACAACATGAAATGCAAGAACGTGGTACCCCTCTATGACCTGCTCCTGGAGATGTTGGATGCCCACCGCCTTCATGCCCCAGCCAGTCGCATGGGAGTGCCCCCAGAGGAGCCCAGCCAGACCCAGCTGGCCACCACCAGCTCCACTTCAGCACATTCCTTACAAACCTACTACATACCCCCGGAAGCAGAGGGCTTCCCCAACACGATCTGA

ADDITIONAL REFERENCES

-   Kicic et al., J Neurosci 23, 7742-7749 (2003).-   Ma et al., J Assoc Res Otolaryngol 1, 129-143 (2000).-   Wang et al., Nature 422, 897-901 (2003).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of generating a differentiated inner earcell, the method comprising: contacting a progenitor cell that expressesone or more of nestin, sox2, musashih, or Pax2, with a gamma secretaseinhibitor in an amount and for a time sufficient to induce theprogenitor cell to express one or more of atonal homolog 1 (Atoh1),jagged 2, p27^(Kip), myosin VIIa, or espin, thereby producing adifferentiated inner ear cell.
 2. The method of claim 1, wherein theprogenitor cell is obtained by a method comprising: providing amesenchymal stem cell; and culturing the mesenchymal stem cell underconditions sufficient to induce the stem cell to differentiate into theprogenitor cell.
 3. The method of claim 2, wherein the mesenchymal stemcell is obtained from a subject who has sensorineural hair cell loss. 4.The method of claim 2, wherein culturing the mesenchymal stem cellcomprises maintaining the stem cell in medium comprising an epidermalgrowth factor, a fibroblast growth factor, or an insulin-like growthfactor.
 5. The method of claim 2, wherein culturing the mesenchymal stemcell comprises maintaining the stem cell in medium comprisingneurotrophin-3 or a brain derived neurotrophic factor.
 6. The method ofclaim 1, wherein the gamma secretase inhibitor is an arylsulfonamide, adibenzazepine, a benzodiazepine,N—[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester(DAPT), L-685,458, or MK0752.
 7. The method of claim 1, wherein thedifferentiated cell is an inner ear auditory hair cell.
 8. The method ofclaim 7, wherein the inner ear auditory hair cell expresses Atoh1,myosin7a, epsin and p²⁷Kip.
 9. The method of claim 1, wherein theprogenitor cell expresses Sox2 and Pax2.
 10. The method of claim 2,wherein the progenitor cell expresses Sox2 and Pax2.